The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides.

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

Proteolysis of human hemoglobin by schistosome cathepsin D.

Schistosomes feed on human blood. They employ proteases to degrade hemoglobin from ingested erythrocytes, using the residues released for amino acid metabolism. However, the identity and the role of the participating protease(s) are unclear and controversial. Confocal microscopy localized schistosomal cathepsin D to the parasite gastrodermis, and revealed elevated protease expression in females. At sub-cellular level, cathepsin D was localized to superficial digestive vacuoles of the gut and to cisternae of the gastrodermal rough endoplasmic reticulum. Schistosome cathepsin D, expressed in insect cells, autoactivated at pH 3.6 to a approximately 40 kDa form that cleaved the substrates o-aminobenzoyl-Ile-Glu-Phe-nitroPhe-Arg-leu-NH(2) and hemoglobin. The NH(2)-terminal residues of mature cathepsin D of Schistosoma japonicum and Schistosoma mansoni were Asn1 and Gly1, respectively, revealing that the proregion peptide was comprised of 35 residues. The proteases cleaved hemoglobin at pH 2.5--4.6, releasing numerous fragments. S. Japonicum cathepsin D cleaved at 13 sites, S. mansoni cathepsin D at 15 sites. Early cleavage sites were alpha Phe33-Leu34 and beta Phe41-Phe42, while others included alpha Leu109-Ala-110 and beta Leu14-Trp15, demonstrating a preference for bulky hydrophobic residues at P1 and P1'. Most of the schistosomal cathepsin D cleavage sites were discrete from those of human cathepsin D. The gastrodermal location, elevated expression in females, acidic pH optima, similar substrate preferences in two species, and the discrete substrate preferences compared with human cathepsin D together provide compelling support for the hypothesis that schistosomal cathepsin D plays an integral role in hemoglobin proteolysis, and might be selectively targeted by drugs based on protease inhibition.

An activated O --> N acyl transfer auxiliary: efficient amide-backbone substitution of hindered "difficult" peptides.

Overcoming the phenomenon known as "difficult" synthetic sequences has been a major goal in solid-phase peptide synthesis for over 30 years. In this work the advantages of amide backbone-substitution in the solid-phase synthesis of "difficult" peptides are augmented by developing an activated N(alpha)()-acyl transfer auxiliary. Apart from disrupting troublesome intermolecular hydrogen-bonding networks, the primary function of the activated N(alpha)()-auxiliary was to facilitate clean and efficient acyl capture of large or beta-branched amino acids and improve acyl transfer yields to the secondary N(alpha)()-amine. We found o-hydroxyl-substituted nitrobenzyl (Hnb) groups were suitable N(alpha)()-auxiliaries for this purpose. The relative acyl transfer efficiency of the Hnb auxiliary was superior to the 2-hydroxy-4-methoxybenzyl (Hmb) auxiliary with protected amino acids of varying size. Significantly, this difference in efficiency was more pronounced between more sterically demanding amino acids. The Hnb auxiliary is readily incorporated at the N(alpha)()-amine during SPPS by reductive alkylation of its corresponding benzaldehyde derivative and conveniently removed by mild photolysis at 366 nm. The usefulness of the Hnb auxiliary for the improvement of coupling efficiencies in the chain-assembly of difficult peptides was demonstrated by the efficient Hnb-assisted Fmoc solid-phase synthesis of a known hindered difficult peptide sequence, STAT-91. This work suggests the Hnb auxiliary will significantly enhance our ability to synthesize difficult polypeptides and increases the applicability of amide-backbone substitution.

Proteolytic degradation of host hemoglobin by schistosomes.

Schistosomes acquire amino acids for growth, development, and reproduction by catabolizing hemoglobin obtained from ingested host erythrocytes. While the biochemical pathway(s) involved has not been determined definitively, a number of proteases including schistosome legumain and cathepsin L-, D-, B- and C-like enzymes have been ascribed roles in the degradation of hemoglobin to diffusible peptides. Transcripts encoding these schistosome proteases, which appear to be expressed in the gastrodermis and cecum of the schistosome, have been reported. Because these enzymes are candidate targets at which to direct novel anti-schistosomal therapies, the comparative biochemistry of these and their counterpart mammalian proteases is now the focus of research in a number of laboratories. This paper reviews reports dating from 40 years ago to the present on how schistosomes digest host-derived hemoglobin, and interprets apparent anomalies in some earlier compared to later reports, the latter having benefited from the availability of PCR and gene cloning technologies. More specifically, the review concentrates on five proteolytic enzymes, and their associated genes, which have been ascribed key roles in the pathway of hemoglobin degradation.

Rational design of potent sialidase-based inhibitors of influenza virus replication.

Two potent inhibitors based on the crystal structure of influenza virus sialidase have been designed. These compounds are effective inhibitors not only of the enzyme, but also of the virus in cell culture and in animal models. The results provide an example of the power of rational, computer-assisted drug design, as well as indicating significant progress in the development of a new therapeutic or prophylactic treatment for influenza infection.